PROJECT SUMMARY The long-term objective of this R21 proposal is to gain a better understanding of the deadly disease of metastatic renal cell carcinoma. Clear cell renal cell carcinoma (ccRCC) is the most common subtype of kidney cancer. Von Hippel Lindau (VHL) is a tumour suppressor that is lost in the majority of ccRCC cases. Loss of VHL stabilizes hypoxia-inducible factors (HIF-1?) protein and upregulates its functional activities. Using the CRISPR genome editing method, we deleted the VHL gene in the murine RENCA and human ACHN ccRCC line and generated novel metastatic ccRCC models. Intriguing findings from our models revealed that VHL gene deletion leads to epithelial mesenchymal transition (EMT) that showed dramatic changes in cell morphology, increased cell motility, and decreased in cell proliferation. Pure clones of VHL-null (VHL-KO) cells grew poorly in vivo. But renal tumors with admixture of VHL-KO and VHL-WT cells exhibited fulminant lung metastases. The outgrowth of lung metastases involves predominantly VHL-WT (non-EMT) cells, suggesting a cooperation between heterogeneous cells is needed for metastasis. This project will undertake major efforts to track down the cooperative interactions between VHL-KO and VHL-WT during in vivo tumor growth and metastasis in both the murine RENCA and human ACHN model. From our recent published study, we have identified HIF-1? and several of its downstream genes, such as periostin, to play key roles in RCC metastasis. The second aim of this project will use the CRISPR genome editing to create compound deletion of VHL and HIF1? and VHL and periostin to verify their role in the metastatic process in cell culture and animal studies. In the third aim, we will examine the expression of VHL, HIF1? and periostin in primary and metastatic RCC clinical specimens to verify their role in RCC aggression and metastasis. Our goal is to gain insights into the molecular mechanism of metastasis which could lead to better treatments for this lethal disease.